Difference between revisions of "Biomod/2013/OSU/protocol/gel prep"

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Latest revision as of 07:29, 26 October 2013


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<a href="http://openwetware.org/wiki/Biomod/2013"> << Back to Team Pages</a>

<img src="http://openwetware.org/images/1/15/Ohiomod2013.jpg" height="60%" width="60%" style="margin-left:-40px;">

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<html>

                       <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/folding_reaction">
<img src="http://openwetware.org/images/9/9e/Protocol_01.gif" width="129" height="125" alt=""></a>

<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/gel_prep">

<img src="http://openwetware.org/images/4/48/Protocol_02.gif" width="99" height="123" border="0" alt=""></a>

<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/negative_staining">

<img src="http://openwetware.org/images/e/ed/Protocol_03.gif" width="116" height="122" border="0" alt=""></a>

<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/custom_tem_grids">

<img src="http://openwetware.org/images/a/ae/Protocol_04.gif" width="93" height="123" border="0" alt=""></a>

<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/drugloading">

<img src="http://openwetware.org/images/e/e9/Protocol_05.gif" width="132" height="123" border="0" alt=""></a>

<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/TIRF">

<img src="http://openwetware.org/images/f/fd/Protocol_06.gif" width="137" height="123" border="0" alt=""></a>

<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/MTT">

<img src="http://openwetware.org/images/2/20/Protocol_07.gif" width="94" height="123" border="0" alt=""></a>
<img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="122" alt="">
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<img src="http://openwetware.org/images/5/59/Protocol_09.gif" width="99" height="2" alt=""> <img src="http://openwetware.org/images/e/e4/Protocol_10.gif" width="211" height="2" alt="">

<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/cellanalysis">

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<img src="http://openwetware.org/images/7/77/Protocol_13.gif" width="108" height="1" alt=""> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="1" alt="">
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Gel Preparation

Prepare a 2% agarose gel (125 ml slab)

  • Weight 2.5 agarose into beaker
  • Fill up to 125 g with 0.5x TBE-Puffer
  • Boil in microwave until agarose is dissolved (2 min)
  • Some water may evaporate through heat. Add ddH2O until back up to 125 grams (apply water to sides of hot beaker)
  • Cool till hand warm
  • Add 1 ml of 1.375 MgCl2 solution
  • Add 7 μL ethidium bromid from 10 mg/ml stock solution. (use gloves for this protocol. minimize exposure)
  • Fill gel tray and install desired comb.
  • Once solid, fill gel box with TBE/11 mM MgCL2 buffer.
  • Remove comb.

Gel Loading

  • Sample: mix 12 μL of each DNA sample with 3 μL 6x loading dye. Load into lanes
  • Unfolded Plasmid: mix 1.2 μL of phage DNA (100 nM) with 10.8 μL ddH2O and 3 μL 6x-loading dye.
  • Load into lanes
  • DNA ladder: add 6 μL of 1kb DNA ladder (New England Biolabs)

Run Gel

  • Put gel box into ice water bath
  • Apply constant 70 V across the gel.
  • Run for 3-4 hours.

Imaging

  • Can image using UV illuminator in dark room. Get picture>exposure>take picture>save under C: share files.

Crunch-n-Squeeze Purification

  • Use UV transilluminator for band visulaization, and cut out the desired band with razor bands.
  • Put slice of gel into tube.
  • Spin down the debris.
  • Cut off debris containing itp of the tub.
  • Invert tip into a freeze'n'squeeze spin column (Biorad).
  • Spin 12 min at 12,000G in tabletop centrifuge.
  • Throw away the top portion of the squeeze spin column.
  • Label and store purified DNA at 4°C

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